Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • A—HUMAN NECESSITIES
  • A21—BAKING; EDIBLE DOUGHS
  • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
  • A21D8/00—Methods for preparing or baking dough
  • A21D8/02—Methods for preparing dough; Treating dough prior to baking
  • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
  • A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
  • A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
  • A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/20—Synthetic spices, flavouring agents or condiments
  • A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
  • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
  • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
  • A23L3/3463—Organic compounds; Microorganisms; Enzymes
  • A23L3/3571—Microorganisms; Enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
  • A61K8/66—Enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/02—Oxygen as only ring hetero atoms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
  • A61K2800/86—Products or compounds obtained by genetic engineering

Definitions

• the invention provides a method of producing hexose oxidase by recombinant DNA technology and such enzyme produced by the method and its use in the food industry and other fields.
• Hexose oxidase (D-hexose:0 2 -oxidoreductase, EC 1.1.3.5) is an enzyme which in the presence of oxygen is capable of oxidiz- ing D-glucose and several other reducing sugars including maltose, lactose and cellobiose to their corresponding lacto- nes with subsequent hydrolysis to the respective aldobionic acids. Accordingly, hexose oxidase differ from another oxido- reductase, glucose oxidase which can only convert D-glucose in that this enzyme can utilize a broader range of sugar substrates.
• the oxidation catalyzed by hexose oxidase can e.g. be illustrated as follows:
• hexose oxidase in the following also referred to as HOX
• HOX hexose oxidase
• isolating the enzyme from several red algal species such as Iridophycus flaccidum (Bean and Hassid, 1956) and Chondrus crispus (Sullivan et al. 1973) .
• the algal species Euthora cristata has been shown to produce hexose oxidase.
• hexose oxidase isolated from these natural sources may be of potential use in the manufacturing of certain food products.
• hexose oxidase isolated from these natural sources may be of potential use in the manufacturing of certain food products.
• Iridophycus flaccidum has been shown to be capable of con- verting lactose in milk with the production of the corres ⁇ ponding aldobionic acid and has been shown to be of potential interest as an acidifying agent in milk, e.g. to replace aci ⁇ difying microbial cultures for that purpose (Rand, 1972) .
• hexose oxidase has been mentioned as a more interesting enzyme than glucose oxidase, since this latter enzyme can only be utilized in milk or food products not containing glucose with the concomitant addition of glucose or, in the case of a milk product, the lactose-degrading enzyme lactase, whereby the lactose is degraded to glucose and galactose. Even if glucose in this manner will become available as a substrate for the glucose oxidase, . it is obvious that only 50% of the end products of lactase can be utilized as substrate by the glucose oxidase, and accordingly glucose oxidase is not an efficient acidifying agent in natural milk or dairy products.
• oxygen oxidoreductases including that of hexose oxidase to generate hydrogen peroxide, which has an antimicrobial effect, has been utilized to improve the stor- age stability of certain food products including cheese, butter and fruit juice as it is disclosed in JP-B-73/016612. It has also been suggested that oxidoreductases may be poten ⁇ tially useful as oxygen scavengers or antioxidants in food products.
• oxidizing agents such as e.g. iodates, peroxides, ascorbic acid, K-bromate or azodicarbonamide for improving the baking performance of flour to achieve a dough with improved stretchability and thus having a desirable strength and stability.
• the mechanism behind this effect of oxidizing agents is that the flour proteins, such as e.g. gluten in wheat flour contains thiol groups which, when they become oxidized, form disulphide bonds whereby the protein forms a more stable matrix resulting in a better dough quality and improvements of the volume and crumb structure of the baked products.
• glucose oxidase as a dough and bread improving additive has the limitation that this enzyme requires the presence of glucose as substrate in order to be effective in a dough system and generally, the glucose con ⁇ tent in cereal flours is low.
• glucose is present in an amount which is in the range of 0-0.4% w/w, i.e. flours may not contain any glucose at all. Therefore, the absence or low content of glucose in doughs will be a limiting factor for the use of glucose oxidase as a dough improving agent.
• the content of maltose is significantly higher already in the freshly prepared dough and further maltose is formed in the dough due to the acti- vity of jS-amylase either being inherently present in the flour or being added.
• the current source of hexose oxidase is crude or partially purified enzyme preparations isolated by extraction from the above natively occurring marine algal species.
• the amount of hexose oxidase in algae is low, it is evident that a production of the enzyme in this manner is too tedious and costly to warrant a cost effective commercial production of the enzyme from these natural sources.
• the provision of a sufficiently pure enzyme product at a cost effective level is not readily achievable in this manner.
• the present invention has, by using recombinant DNA techno ⁇ logy, for the first time made it possible to provide hexose oxidase active polypeptides in industrially appropriate quantities and at a quality and purity level which renders the hexose oxidase active polypeptide according to the inven ⁇ tion highly suitable for any relevant industrial purpose including the manufacturing of food products and pharmaceuti ⁇ cals.
• the invention pertains in a first aspect to a method of producing a polypeptide having hexose oxidase activity, comprising isolating or synthesizing a DNA fragment encoding the polypeptide, introducing said DNA fragment into an appropriate host organism in which the DNA fragment is combined with an appropriate expression signal for the DNA fragment, cultivating the host organism under conditions leading to expression of the hexose oxidase active polypeptide and recovering the polypeptide from the cultiva ⁇ tion medium or from the host organism.
• the invention relates to a polypeptide in isolated form having hexose oxidase activity, comprising at least one amino acid sequence selected from the group consisting of
• X represents an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Asx, Cys, Gin, Glu, Glx, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, and muteins and variants hereof.
• the invention relates to a recombi ⁇ nant DNA molecule comprising a DNA fragment coding for a polypeptide having hexose oxidase activity and to a microbial cell comprising such a recombinant DNA molecule.
• the invention pertains to the use of the above hexose oxidase active polypeptide or a microbial cell expressing such a polypeptide in the manufacturing of a food product or an animal feed and the manufacturing of a pharma ⁇ ceutical product, a cosmetic or a tooth care product.
• a method of reduc ⁇ ing the sugar content of a food product comprising adding to the food product an amount of the polypeptide or the icro- bial cell as disclosed herein, which is sufficient to remove at least part of the sugar initially present in said food product, a method of preparing a baked product from a dough, comprising adding the hexose oxidase active polypeptide or a microbial cell expressing such a polypeptide to the dough, and a dough improving composition comprising the polypeptide or the microbial cell according to the invention and at least one conventional dough component.
• the invention relates to the use of the polypeptide or a microbial cell according to the invention as an analytical reagent for measuring the content of sugars.
• the invention also provides the use of a polypeptide or a microbial cell according to the inven ⁇ tion in the manufacturing of a lactone, whereby the polypeptide and/or the microbial cell is applied to a reactor containing a carbohydrate which can be oxidized by the polypeptide and operating the reactor under conditions where the carbohydrate is oxidized.
• Hexose oxidases are produced naturally by several marine algal species. Such species are i.a. found in the family Gigartinaceae which belong to the order Gigartinales . Examples of hexose oxidase producing algal species belonging to Gigartinaceae are Chondrus crispus and Iridophycus flacci ⁇ dum. Also algal species of the order Cryptomeniales including the species Euthora cristata are potential sources of the hexose oxidase active polypeptide according to the invention. Accordingly, such algal species are potentially useful sources of hexose oxidase and of DNA coding for such hexose oxidase active polypeptides.
• hexose oxidase active polypeptide denotes an enzyme which at least oxidizes D-glucose, D-galactose, D-mannose, maltose, lactose and cellobiose.
• the enzyme is typically isolated from the algal starting material by extraction using an aqueous extraction medium.
• the algae may be used in their fresh state as harvested from the marine area of growth or they may be used after drying the fronds e.g. by air-drying at ambient temperatures or by any appropriate industrial drying method such as drying in circulated heated air or by freeze-drying.
• the fresh or dried starting material may be comminuted e.g. by grinding or blending.
• buffer solutions having a pH in the range of 6-8, such as 0.1 M sodium phosphate buf ⁇ fer, 20 mM triethanolamine buffer or 20 mM Tris-HCl buffer are suitable.
• the hexose oxidase is typically extracted from the algal material by suspending the starting material in the buffer and keeping the suspension at a temperature in the range of 0-20°C such as at about 5°C for 1 to 10 days, pre- ferably under agitation.
• the suspended algal material is then separated from the aqueous medium by an appropriate separation method such as filtration, sieving or centrifugation and the hexose oxidase subsequently recovered from the filtrate or supernatant.
• an appropriate separation method such as filtration, sieving or centrifugation and the hexose oxidase subsequently recovered from the filtrate or supernatant.
• the separated algal material is subjected to one or more further extraction steps.
• the pigments may be removed by treating the filtrate or supernatant with an organic solvent in which the pigments are soluble and subsequently separating the solvent containing the dissolved pigments from the aqueous medium.
• the recovery of hexose oxidase from the aqueous extraction medium can be carried out by any suitable conventional methods allowing isolation of proteins from aqueous media. Such methods, examples of which will be described in details in the following, include such methods as ion exchange chro- matography, optionally followed by a concentration step such as ultrafiltration. It is also possible to recover the enzyme by adding substances such as e.g. (NH 4 ) 2 S0 4 which causes the protein to precipitate, followed by separating the precipi ⁇ tate and optionally subjecting it to conditions allowing the protein to dissolve.
• substances such as e.g. (NH 4 ) 2 S0 4 which causes the protein to precipitate, followed by separating the precipi ⁇ tate and optionally subjecting it to conditions allowing the protein to dissolve.
• the enzyme in a substantially pure form e.g. as a preparation essentially without other proteins or non-protein contaminants and accordingly, the relatively crude enzyme preparation resulting from the above extraction and isolation steps is preferably subjected to further purification steps such as further chromatography steps, gel filtration or chromatofocusing as it will also be described by way of example in the following.
• the hexose oxidase active polypeptide according to invention is provided by means of recombinant DNA technology methods allowing it to be produced by cultivating in a culturing medium an appropriate host organism cell comprising a gene coding for the hexose oxidase, and recovering the enzyme from the cells and/or the culturing medium.
• the method of producing hexose oxidase which is provided herein comprises as a first step the isolation or the con- struction of a DNA fragment coding for hexose oxidase.
• Se ⁇ veral strategies for providing such a DNA fragment are avail ⁇ able.
• the DNA fragment can be isolated as such from an organism which inherently produces hexose oxidase.
• it is required to dispose of RNA or DNA probe sequences which under appropriate conditions will hybridize to the DNA fragment searched for and subsequently isolating a DNA fragment com ⁇ prising the coding sequence and cloning it in a suitable cloning vector.
• Another suitable strategy which is disclosed in details in the below examples, is to isolate mRNA from an organism producing the hexose oxidase and use such mRNA as the start ⁇ ing point for the construction of a cDNA library which can then be used for polymerase chain reaction (PCR) synthesis of DNA based on oligonucleotide primers which are synthesized based on amino acid sequences of the hexose oxidase. It was found that such a strategy is suitable for providing a hexose oxidase-encoding DNA fragment. By way of example such a strategy as described in details below is described summar- ily.
• Synthetic oligonucleotides were prepared based on the HOX-2 and HOX-3 peptide sequences prepared as described hereinbelow by endoLys-C digestion of a 40 kD polypeptide of hexose oxidase extracted from Chondrus crispus .
• PCR using first strand cDNA as template and with a sense HOX-2 primer and an anti-sense HOX-3 primer produced a DNA fragment of 407 bp. This fragment was inserted into an E. coli vector, pT7 Blue and subsequently sequenced.
• this 407 bp fragment also contained an open reading frame containing the HOX-4 and HOX-5 peptides of the above 40 kD Chondrus crispus- derived hexose oxidase fragment the isolation of .which is also described in the following.
• Sense and anti-sense oligonucleotides were synthesized based on the 407 bp fragment, and two fragments of 800 and 1400 bp, respectively could subsequently be isolated by PCR using cDNA as template. These two fragments were cloned in the pT7 Blue vector and subsequently sequenced. The DNA sequence of the 5' -fragment showed an open reading frame containing the HOX-6 peptide which was also isolated from the above 40 kD Chondrus crispus-derived hexose oxidase fragment.
• the 3'- fragment showed a reading frame containing the HOX-1, the isolation of which is disclosed below, and the HOX-7 and HOX-8, both isolated from a 29 kD Chondrus crispus-derived hexose oxidase polypeptide obtained by endoLys-C digestion as also described in the following.
• an oligonucleotide corresponding to the 5' -end of the presumed hox gene and an oligonucleotide corresponding to the 3' -end of that gene were synthesized. These two oligonucleotides were used in PCR using first strand cDNA as template result ⁇ ing in a DNA fragment of about 1.8 kb. This fragment was cloned in the above E. coli vector and sequenced.
• the DNA sequence was identical to the combined sequence of the above 5' -end, 407 bp and 3'-end sequences and it was concluded that this about 1.8 kb DNA sequence codes for both the 40 kD and the 29 kD Chondrus crispus-derived hexose oxidase fragments.
• the above strate ⁇ gy for isolating a DNA fragment encoding a hexose oxidase active polypeptide can be used for the construction of such fragments encoding hexose oxidases derived from any other natural source than Chondrus crispus including the marine algal species mentioned above, such as from other plants or from a microorganism.
• the DNA sequence of the hexose oxidase active polypeptide-encoding DNA fragment may be constructed syn ⁇ thetically by established standard methods e.g. the phospho- amidite method described by Beaucage and Caruthers (1981) , or the method described by Matthes et al. (1984).
• phosphoamidite method oligonucleotides are synthesized, eg in an automatic DNA synthesizer, purified, annealed, ligated and cloned in an appropriate vector.
• the DNA fragment may be of mixed genomic and synthetic, mixed synthetic and cDNA or mixed genomic and cDNA origin, prepared by ligating sub-fragments of synthetic, genomic or cDNA origin as appropriate, the sub-fragments corresponding to various parts of the entire DNA fragment, in accordance with standard techniques.
• the isolated or synthesized hexose oxidase active polypeptide-encoding DNA fragment is introduced into an appropriate host organism in which the DNA fragment is com ⁇ bined operably with an appropriate expression signal for the DNA fragment.
• an introduction can be carried out by methods which are well-known to the skilled practitioner including the construction of a vector having the fragment inserted and transforming the host organism with the vector. Suitable vectors include plasmids which are capable of repli- cation in the selected host organism. It is also contemplated that the DNA fragment can be integrated into the chromosome of the host organism e.g. by inserting the fragment into a transposable element such as a transposon, and subjecting a mixture of the selected host organism and the transposon to conditions where the transposon will become integrated into the host organism chromosome and combine with an appropriate expression signal.
• a hexose oxidase active polypeptide-encoding DNA fragment including the gene for the polypeptide which is produced by methods as described above, or any alternative methods known in the art, can be expressed in enzymatically active form using an expression vector.
• An expression vector usually includes the components of a typi- cal cloning vector, i.e. an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypic markers for selection purposes.
• An expression vector includes control sequences encoding a promoter, operator, ribosome binding site, translation initi- ation signal and optionally, a repressor gene or one or more activator genes.
• a signal sequence may be inserted upstream of the coding sequence of the gene.
• expression signal includes any of the above control sequences, repressor or activator sequences and signal sequence.
• the hexose oxidase encoding gene is operably linked to the control sequences in proper manner with respect to expression.
• Promoter sequences that can be incorporated into plasmid vectors, and which can support the transcription of the hexose oxidase gene include, but are not limited to the tac promoter, phage lambda-derived promoters including the P L and P R promoters.
• An expression vector carrying the DNA fragment of the inven- tion may be any vector which is capable of expressing the hexose oxidase gene in the selected host organism, and the choice of vector will depend on the host cell into which it is to be introduced.
• the vector may be an autonomously replicating vector, i.e. a vector which exists as an extra- chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid, a bacteriophage or an extrachromosomal element, a minichromosome or an arti ⁇ ficial chromosome.
• the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromo- some.
• the DNA fragment coding for the hexose oxidase active polypeptide should be operably combined with a suit ⁇ able promoter sequence.
• the promoter may be any DNA sequence which confers transcriptional activity to the host organism of choice and may be derived from genes encoding proteins which are either homologous or heterologous to the host organism. Examples of suitable promoters for directing the transcription of the DNA fragment of the invention in a bacterial host are the promoter of the lac operon of E.
• the Streptomyces coelicolor agarase gene dagrA promoters the promoters of the Bacillus licheniformis o.-amylase gene (amyL) , the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM) , the promoters of the Bacillus amyloliquefaciens ⁇ -amylase gene (a yQ) , the promoters of the Bacillus subtilis xylA and xylB genes.
• examples of useful promoters are those derived from the genes encoding the Pichia pastoris alcohol oxidase, Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral o.-amylase, A. niger acid stable o.-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase or Aspergillus nidulans acetamidase.
• Suitable promoters for expression in a yeast species the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae can be mentioned.
• a suitable promoter When expressed in a bacterial species such as E. coli , a suitable promoter may be selected from a bacteriophage promoter including a T7 promoter or a lambda bacteriophage promoter.
• the vector comprising the DNA fragment encoding the hexose oxidase active polypeptide may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host organism such as a mutation conferring an auxo- throphic phenotype, or the marker may be one which confers antibiotic resistance or resistance to heavy metal ions.
• a selectable marker e.g. a gene the product of which complements a defect in the host organism such as a mutation conferring an auxo- throphic phenotype, or the marker may be one which confers antibiotic resistance or resistance to heavy metal ions.
• the host organism of the invention either comprising a DNA construct or an expression vector as described above is advantageously used as a host cell in the recombinant produc ⁇ tion of a polypeptide according to the invention.
• the cell may be transformed with a DNA construct comprising the gene coding for the polypeptide of the invention or, conveniently by integrating the DNA construct into the host chromosome.
• Such an integration is generally considered to be advantage ⁇ ous as the DNA fragment is more likely to be stably main ⁇ tained in the cell.
• Integration of the DNA constructs into the host chromosome may be carried out according to conven ⁇ tional methods such as e.g. by homologous or heterologous recombination or by means of a transposable element.
• the host organism may be transformed with an expres ⁇ sion vector as described above.
• the host organism may be a cell of a higher organism such as an animal cell, including a mammal, an avian or an insect cell, or a plant cell.
• the host organism is a microbial cell, e.g. a bacterial or a fungal cell including a yeast cell.
• suitable bacterial host organisms are gram posi ⁇ tive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis , Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus , Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium and Bacillus thuringiensis , Streptomyces species such as Streptomyces murinus, lactic acid bacterial species including Lactococcus spp. such as Lactococcus lactis, Lactobacillus spp.
• Bacillaceae including Bacillus subtilis, Bacillus licheniformis , Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus , Bacillus amyloliquefaciens, Bacillus coagulans
• strains of a gram negative bacterial species such as a species belonging to Enter obacteriaceae, including E. coli or to Pseudomonadaceae may be selected as the host organism.
• a yeast host organism may advantageously be selected from a species of Saccharomyces including Saccharomyces cerevisiae or a species belonging to Schizosaccharo yces. Suitable host organisms among filamentous fungi include species of Asper ⁇ gillus, eg Aspergillus oryzae, Aspergillus nidulans or Asper ⁇ gillus niger. Alternatively, strains of a Fusarium species, eg Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. In one preferred embodiment a strain of the species Pichia pastor is is used as host organism.
• Some of the above useful host organisms such as fungal spe ⁇ cies or gram positive bacterial species may be transformed by a process which include protoplast formation and transfor- mation of the protoplasts followed by regeneration of the cell wall in a manner known per se.
• the recombinant host organism cells as described above are cultivated under conditions which lead to expression of the polypeptide in a recoverable form.
• the medium used to cul ⁇ tivate the cells may be any conventional medium suitable for growing the host cells in question and obtaining expression of the polypeptide. Suitable media are available from commer ⁇ cial suppliers or may be prepared according to published recipes.
• the resulting polypeptide is typically recovered from the cultivation medium by conventional procedures including separating the cells from the medium by centrifugation or filtration, if necessary, after disruption of the cells, followed by precipitating the proteinaceous components of the supernatant or filtrate e.g. by adding a salt such as ammon ⁇ ium sulphate, followed by a purification step.
• microbial cultures such as e.g. bacterial cultures which are used in the manufacturing of food or feed products can be used as the host organism expressing the gene coding for the hexose oxidase active polypeptide.
• lactic acid bacteri ⁇ al starter cultures which are used in the manufacturing of dairy products or other food products such as meat product or wine and which e.g. comprise one or more strains of a lactic acid bacterium selected from any of the above lactic acid bacterial species can be used as host organisms whereby the hexose oxidase will be produced directly in the food product to which the starter cultures are added.
• the hexose oxidase encoding gene according to the invention may be introduced into lactic acid bacterial starter cultures which are used as inoculants added to fodder crops such as grass or corn or to proteinaceous waste pro ⁇ ducts of animal origin such as fish and slaughterhouse waste materials for the production of silage for feeding of ani ⁇ mals.
• the expression of hexose oxidase by the silage inoculants will imply that the oxygen initially present in the crops or the waste materials to be ensiled is depleted whereby anaerobic conditions, which will inhibit growth of aerobic spoilage organisms such as gram negative bacteria and yeasts, will be established.
• yeast cultures such as baker's yeast or yeast cultures which are used in the manufacturing of alcoholic beverages including wine and beer can be used as host organisms for the gene coding for the hexose oxidase active polypeptide of the invention.
• yeast cultures such as baker's yeast or yeast cultures which are used in the manufacturing of alcoholic beverages including wine and beer can be used as host organisms for the gene coding for the hexose oxidase active polypeptide of the invention.
• the hexose oxidase being produced will have a dough improving effect as it is described in the following.
• the recombi ⁇ nant microbial cultures expressing a hexose oxidase active polypeptide are used in a bioreactor for the production of the enzyme or for the production of lactones from either of the above-mentioned carbohydrates which can be oxidized by the hexose oxidase active enzyme.
• the cells of the microbial cultures are advantageously immobilized on a solid support such as a polymer material, which is preferable in the form of small particles to provide a large surface for binding the cells.
• the isolated enzyme may be used for the above purpose, also preferable bound to a solid support material.
• the binding of the cells or the enzyme may be provided by any conventional method for that purpose.
• the polypeptide having hexose oxidase activity may be a fusion product, i.e. a polypeptide which in addition to the hexose oxidase active amino acid sequences comprises further amino acid sequences having other useful activities.
• fusion polypeptides having one or more enzyme activities in addition to the hexose oxidase activity are contemplated.
• Such additional enzyme activities may be chosen among enzymes capable of degrading carbohydrates, such as lactase, amylases including glucoamylases, glucanases, cellulases, hemicellulases, xyla- nases, lactases or any other oxidoreductase such as glucose oxidase, galactose oxidase or pyranose oxidase, and also among proteases and peptidases, lipases or nucleases.
• the additional enzyme sequence(s) to be chosen for integration into a hexose oxidase polypeptide according to the invention depend(s) on the product for which the enzymatically active fusion product is intended.
• a hexose oxidase active fusion polypeptide for use in the manufacturing of a dairy product advantageously comprises a lactase, a protease or a peptidase, and that a fusion polypeptide intended for dough improvement may as the fusion partner comprise any of the above carbohydrate degrad ⁇ ing enzymes.
• microbial cells accord ⁇ ing to the invention as described above and which express a hexose oxidase active fusion polypeptide having additional enzyme activities may be used for inoculation of other food products and animal feeds in the manner as also described above.
• a suitable fusion partner may be a sequence conferring to the hexose oxidase altered charac ⁇ teristics such as solubility or a sequence which can serve as a "tagging" group conferring to the hexose oxidase the abi ⁇ lity to bind more strongly or more selectively to a particu ⁇ lar solid material for hexose oxidase polypeptide purifica ⁇ tion or immobilization purposes.
• polypeptide as a chimeric product comprising partial sequences of hexose oxidase active polypeptides derived from different sources and being encoded by a DNA fragment which is constructed by combining hexose oxidase active polypeptide-encoding DNA sequences from these diffe- rent sources into one DNA fragment encoding the entire chi ⁇ meric polypeptide.
• the method according to the inven ⁇ tion is one wherein the DNA fragment encoding the hexose oxidase active polypeptide comprises at least one DNA sequence coding for an amino acid sequence selected from the group consisting of
• X represents an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Asx, Cys, Gin, Glu, Glx, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val,
• the term "variant" is used to desig ⁇ nate any modification of a hexose oxidase active polypeptide sequence which does not result in complete loss of the hexose oxidase activity.
• the modifications may include deletion, substitution of amino acid residues present in the polypeptide as it is derived from a natural source or in an already modified polypeptide sequence or the modification may imply the insertion into such a polypeptide of additional amino acid residues.
• Substitution of one or more amino acid residues may be carried out by modifying or substituting the codon or codons coding for the amino acid or amino acids which it is desired to substitute, e.g.
• deletion of one or more amino acid residues can be made by deleting the corresponding codon or codons in the DNA fragment coding for the polypeptide accord- ing to the invention.
• the method according to .the inven ⁇ tion may as a further step include a purification of the polypeptide preparation initially recovered from the cultiva ⁇ tion medium and/or the microorganisms.
• the purpose of this further step is to obtain an enzyme preparation in which the hexose oxidase polypeptide is in a substantially pure form.
• substantially pure form implies that the prepara ⁇ tion is without any undesired contaminating substances origi ⁇ nating from the cultivation medium, the production host organism cells or substances produced by these cells during cultivation.
• the polypeptide preparation resulting from the purifica ⁇ tion step is substantially without any non-hexose oxidase enzymatic activity.
• the purification methods will depend on the degree of purity which is desirable, but will typically be selected from conventional protein purification methods such as salting out, affinity or ion exchange chromatography procedures including hydrophobic interaction chromatography, and gel filtration methods, such as the method described in the following examples.
• the invention relates in a further aspect to a polypeptide in isolated form having hexose oxidase activity, comprising at least one of the above amino acid sequences, or muteins and variants hereof as they are described above.
• the polypeptide is produced according to the methods as described above.
• the polypeptide according to the inven ⁇ tion may be glycosylated to a varying degree or it may for certain purposes advantageously be expressed in a substan- tially non-glycosylated form.
• the polypeptide is one which has functional characteristics identical or partially identical to those of hexose oxidase naturally occurring in the algal species Chondrus crispus as they are described in the prior art. It was found that such a hexose oxidase extracted from the algal source when it was subjected to SDS-PAGE as described herein may show separate protein bands of 29, 40 and/or 60 kD.
• the enzyme has a high enzymatic activity over a broad pH range.
• the hexose oxidase according to the invention at least shows an enzymatic activity at a pH in the range of 1- 9, such as in the range of 2-9 including the range of 5-9.
• the pH range of activity or the pH optimum of a naturally derived hexose oxidase may be modified in a desired direction and to a desired degree by modifying the enzyme as described above or by random mutagenesis of a replicon or a host organism com- prising the DNA coding for the hexose oxidase, followed by selection of mutants having the desired altered pH charac ⁇ teristics.
• modifications of the enzyme may be aimed at modifying the thermotolerance and optimum temperature for activity of the hexose oxidase active polypeptide, or at changing the isoelectric point of the enzyme.
• the polypeptide according to the invention is preferably enzymatically active within a broad temperature range such as a range of 10-90°C, e.g. within a range of 15- 80°C including the range of 20-60°C.
• a broad temperature range such as a range of 10-90°C, e.g. within a range of 15- 80°C including the range of 20-60°C.
• the hexose oxidase active polypeptide maintains a significant residual enzymatic activity at temperatures of 70°C or higher, e.g. when the enzyme is intended for use in doughs where it may be useful to have hexose oxidase activity during at least part of the subsequent baking step.
• hexose oxidase depends on the range of carbohydrates which they can use as substrate. Although the hexose oxidase appear to have highest substrate specificity for hexoses, such as glucose, galactose and mannose, it has been found that the range of carbohydrate substances which can be utilized as substrates for the polypeptide according to the invention is not limited to hexoses.
• a preferred polypeptide is one which in addi- tion to a high specificity for hexoses also has a high speci ⁇ ficity for other carbohydrate substances including disaccharides such as lactose, maltose and/or cellobiose and even also substantial specificity to pentoses including as an example xylose, or deoxypentoses or deoxyhexoses such as rhamnose or fucose.
• hexose oxidase in addition to a high specifi ⁇ city to hexoses and other monosaccharides also has substan ⁇ tial specificity for disaccharides, in particular lactose present in milk and maltose which i.a. occurs in cereal flours and doughs.
• polypeptide according to the invention is one which in addition to D-glu- cose oxidizes at least one sugar selected from the group consisting of D-galactose, maltose, cellobiose, lactose, D-mannose, D-fucose and D-xylose.
• the hexose oxidase active polypeptide has an isoelectric point in the range of 4-5.
• the polypeptide may preferably have an isoelectric point of 4.3 ⁇ 0.1 or one of 4.5 ⁇ 0.1.
• the polypeptide according to the invention typi ⁇ cally has a molecular weight as determined by gel filtration using Sephacryl S-200 Superfine (Pharmacia) which is in the range of 100-150 kD, A molecular weight determined by this or equivalent methods are also referred to as an apparent mole ⁇ cular weight.
• the polypeptide may have an apparent molecular weight of 110 kD ⁇ 10 kD.
• the invention provides a recombi ⁇ nant DNA molecule comprising a DNA fragment coding for a polypeptide having hexose oxidase activity.
• a DNA fragment may be isolated from a natural source or it may be constructed e.g. as it is described in details in the below examples.
• the coding fragment may also be synthesized based upon amino acid sequences of a naturally occurring hexose oxidase.
• the recom ⁇ binant molecule can be selected from any of the expression vector types as mentioned above.
• the recombinant DNA molecule comprises a DNA fragment coding for a hexose oxidase polypeptide which comprises at least one of the above amino acid sequences (i) to (viii) , or a mutein or derivative of such polypeptide.
• the recombinant DNA molecule comprises the following DNA sequence (SEQ ID NO:30) :
• CTCAAGGTGC TTGTACTGCA CTTGACCGTG CTATGGAAAA GTGTTCTCCC GGTACAGTCA 300
• TCATCAACGT CACTGGTCTC GTTGAGAGTG GTTATGACGA CGATAGGGGT TACTTCGTCA 420
• GGCACACATC CAAGACGTCG TATATGCATG ACGAGACGAT GGACTACCCC TTCTACGCGC 1140
• GCATCCCTAC TAAACCTCTT AAGGAGCCCA AGCAGACGAA ATAGTAGGTC ACAATTAGTC 1740
• the invention provides in another aspect a microbial cell which comprises the above recombinant DNA molecule.
• the above general description of the host organism comprising a DNA fragment encoding the polypeptide according to the invention encompasses such a microbial cell and accor ⁇ dingly, such cells can be selected from any of the above mentioned microbial groups, families, genera and species, i.e. the microbial cell may be selected from a bacterial cell, a fungal cell and a yeast cell including as examples an E. coli cell, a lactic acid bacterial cell, a Saccharomyces cerevisiae cell and a Pichia pastoris cell.
• the microbial cell according to the invention may, if it is intended for direct addition to a product where it is desired to have hexose oxidase activity, e.g. during a manufacturing process, be provided in the form of a microbial culture, preferable in a concentrate form.
• a culture may advantageously contain the microbial cell according to the invention in a concentration which is preferably in the range of 10 5 to 10 12 per g of culture.
• the culture may be a fresh culture, i.e. a non-frozen suspension of the cells in a liquid medium or it may in the form of a frozen or dried culture, e.g. a freeze-dried culture.
• the microbial cell may also for specific purposes be immobilized on a solid substrate.
• the invention relates in another further aspect to the use of the hexose oxidase active polypeptide according to the invention or of a microbial cell expressing such a polypeptide in the manufacturing of food products.
• the term "manufacturing" should be understood in its broadest sense so as to encompass addition of the hexose oxidase or the microbial cell to ingredients for the food product in question, prior to, during or after any subsequent process step, during packaging and during storage of the finished product up till it is consumed.
• the food products where such use is advantageous may be any product where the end products of the hexose oxidase confer advantageous effects on the food product.
• the desired activity of the hexose oxidase will only be obtained if substrate for the enzyme is present in sufficient amounts.
• the substrate carbohydrates may be inhe ⁇ rently present in the food product or the ingredients herefor or they may be added or generated during the manufacturing process.
• An example of substrate being generated during manufacturing is the enzymatic degradation of di-, oligo- or polysaccharides to lower sugar substances which is degradable by the hexose oxidase which may occur as the result of enzy ⁇ matic activity of enzymes inherently present in the food product or added during the manufacturing.
• substrate for the hexose oxidase active polypeptide may be generated as the result of the enzymatic activity of a fusion partner as described above.
• hexose oxidase activity in a product containing substrates for the enzyme include generation of lactones from the sugar substrate which may subsequently be converted to corresponding acids, generation of hydrogen peroxide and consumption of oxygen.
• Typical examples of food products where hexose oxidase acti ⁇ vity may be advantageous include as examples dairy products, starch-containing food products and non-dairy beverages.
• the consumption of oxygen resulting from the activity of the hexose oxidase has several advantageous implications in relation to the manufacturing of food products and pharmaceu ⁇ ticals.
• the hexose oxidase may act as an antioxidant and additionally, the reduction of oxygen content may inhibit spoilage organisms the growth of which is depen ⁇ dent on presence of oxygen and accordingly, the hexose oxidase active polypeptide may also act as an antimicrobial agent.
• hexose oxidase active polypeptide accor ⁇ ding can be utilized to extend the shelf life of packaged foods where spoilage can be prevented by the incorporation of the hexose oxidase active polypeptide accor ⁇ ding to the invention either in the food product itself or by providing a mixture of the hexose oxidase and an appropriate substrate herefor in the packaging, but separate from the content of food product.
• a mixture is attached to the inner side of a food container such as eg a tin or a jar.
• the hexose oxidase according to the invention can be used as an oxygen removing agent in a food packaging.
• the hexose oxidase according to the inven ⁇ tion is useful as oxygen removing and acidifying agent in the ensiling of feeds, optionally in the form of compositions further comprising one or more conventional silage additive such as lactic acid bacterial inoculants or enzymes which generate low molecular sugar substances.
• a further useful application of the hexose oxidase polypeptide according to the invention is the use of the enzyme to reduce the sugar content of a food product, com- prising adding to the product an amount of the polypeptide or a microbial cell producing the polypeptide which is suffi ⁇ cient to remove at least part of the sugar initially present in the food product.
• Such an application may e.g. be useful in the manufacturing of diets for diabetic patients where a low sugar content is desired, and in the production of wines with a reduced alcohol content.
• the hexose oxidase is preferably added to the must prior to yeast inoculation.
• the invention relates to the use of the hexose oxidase active polypeptide or of a .microbial cell producing the enzyme according to the invention in the manufacturing of pharmaceutical products, cosmetics or tooth care products such as tooth pastes or dentrifices.
• the desired effects of the hexose oxidase in such products are essentially those described above with respect to food pro ⁇ ducts and animal feeds.
• hexose oxidase accor ⁇ ding is its use as a dough improving agent. It has been found that the addition of the hexose oxidase to a dough results in an increased resistance hereof to breaking when the dough is stretched, i.e. the enzyme confers to the dough an increased strength whereby it becomes less prone to mechanical deformation.
• this effect of addition of the hexose oxidase according to the invention to a dough is the result of the formation of cross ⁇ links between thiol groups in sulphur-containing amino acids in flour proteins which occurs when the hydrogen peroxide generated by the enzyme in the dough reacts with the thiol groups which are hereby oxidized.
• the invention also provides a method of prepar ⁇ ing a baked product from a dough, comprising adding to the dough an effective amount of the polypeptide or a microorga- nism according to the invention which is capable of expres- sing such a polypeptide, and a dough improving composition comprising the polypeptide or a microorganism capable of expressing such a polypeptide in a dough, and at least one conventional dough component.
• a dough improving composition comprising the polypeptide or a microorganism capable of expressing such a polypeptide in a dough, and at least one conventional dough component.
• such a composition may further comprise at least one dough or bakery product improving enzyme e.g.
• a cellulase selected from a cellulase, a hemicellulase, a pentosanase, a lipase, a xylanase, an amylase, a glucose oxidase and a protease.
• the hexose oxidase is used as an analytical reagent in methods of determining in a biological and other samples the concentration of any sugar which can be converted by the enzyme.
• the sugar content is measured by determining the amount of end products resulting from the enzymatic conversion of the substrate sugar present in the sample to be measured.
• the hexose oxidase can be used directly as a reagent in an in vitro analytical assay or that it can be incorporated in a sensor.
• Figure 1 represents a schematic overview of the purification of hexose oxidase (HOX) and the two strategies adopted for obtaining amino acid sequence information
• Figure 2 shows native, non-dissociating polyacrylamide gel electrophoresis (native PAGE) of preparations of hexose oxidase at different steps of the purification.
• the samples represent the enzyme preparation obtained after anion exchange chromatography and concentration (lane 1) , after gel filtration (lane 2) , and after either cation exchange chroma- tography (lane 3) or chromatofocusing (lane 4) .
• the Phast gel (Pharmacia, 8-25% gradient gel) was silver stained. Molecular weights of standard proteins (x 10 ⁇ 3 ) are indicated at the left.
• the band corresponding to hexose oxidase which is indicated by an arrow, was identified by enzyme staining of another gel in parallel (not shown) .
• the four lanes were run on separate gels,
• Figure 3 shows the UV-profile obtained during purification of hexose oxidase by gel filtration on Sephacryl S-200 HR as described in the text. Fractions containing hexose oxidase (HOX) activity are indicated by the filled area,
• Figure 4 shows SDS-PAGE of hexose oxidase purified from Chondrus crispus by anion exchange chromatography on DEAE- Sepharose Fast Flow, gel filtration on Sephacryl S-200, followed by either cation exchange chromatography on S-Sepha- rose Fast Flow (lane 1) or chromatofocusing on a Mono P column (lane 2) .
• Molecular weights of standard proteins xlO "3 ) are indicated at the left.
• the polypeptides at 60 kD, 40 kD and 29 kD are marked by arrows.
• Reduced samples were run on a 12% polyacrylamide gel which was stained with Coo- massie Brilliant Blue R-250. The two lanes were run on sepa ⁇ rate gels,
• Figure 5 shows isoelectric focusing (IEF) of hexose oxidase.
• the gel was either stained with Coomassie Brilliant Blue R- 250 (lane 1) or stained for enzyme activity as described in the text (lane 2) .
• the positions of isoelectric point markers run in parallel are shown at the left. The two lanes were run on separate gels,
• Figure 6 shows reversed phase HPLC separation of peptides generated by digestion of the 40 kD HOX-polypeptide with endoproteinase Lys-C.
• the peaks labelled 1, 2, 3, 4 and 5 were subjected to amino acid sequencing,
• Figure 7 shows reversed phase HPLC separation of peptides generated by digestion of the 29 kD HOX-polypeptide with endoproteinase Lys-C.
• the peaks labelled 1 and 2 were sub ⁇ jected to amino acid sequencing
• Figure 8 shows a Northern blot analysis of RNA extracted from Chondrus crispus.
• the denaturing agarose gel was loaded with 30 ⁇ g (lane 1) and 3 ⁇ g (lane 2) , respectively of total RNA.
• Left arrow indicates hexose oxidase specific transcript.
• the positions of molecular weight markers in kb are shown to the right,
• Figure 9 shows the construction of plasmid pUP0153 which mediates the expression of recombinant hexose oxidase in Pichia pastoris. Small arrows indicate PCR primers. The grey box indicates the hexose oxidase gene,
• Figure 10 shows purification of recombinant hexose oxidase from Pichia pastoris by anion exchange chromatography on HiTrap-Q column (step one) .
• Alcohol oxidase (AOX) activity (•) and hexose oxidase (HOX) activity (o) in the collected fractions were assayed as described in the text,
• Figure 11 shows purification of recombinant hexose oxidase from Pichia pastoris by gel filtration on Sephacryl S-200 HR (step two) .
• Alcohol oxidase (AOX) activity (•) and hexose oxidase (HOX) activity (o) in the collected fractions were assayed as described in the text,
• Figure 12 shows the construction of plasmid pUPOl ⁇ l which mediates the expression of recombinant hexose oxidase in E. coli .
• Small arrows indicate PCR primers (grey box indicates the hexose oxidase gene) ,
• Figure 13 shows SDS-PAGE of recombinant hexose oxidase pro ⁇ quizd in E. coli .
• Crude extracts from lysed cells were ana ⁇ lyzed in a 14% denaturing gel.
• Molecular weights of standard proteins x 10 "3 ) are indicated to the left.
• the gel was stained with Coomassie Brilliant Blue R-250.
• Lane 1 shows extract from E. coli cells with pUPOl ⁇ l
• lane 2 shows plasmid-less control.
• Figure 14 shows the construction of plasmid pUP0155 which mediates the expression of recombinant hexose oxidase in Saccharomyces cerevisiae. Small arrows indicate PCR primers. The grey box indicates the hexose oxidase gene.
• FIG. 1 A schematic overview of the purification and two strategies adopted for obtaining amino acid sequence information for the enzyme is shown in Fig. 1.
• the red sea-weed Chondrus crispus was collected during April to September at the shore near Grena, Jutland, Denmark at a depth of 2-5 meters. Freshly collected algal fronds were rinsed with cold water and stored on ice during transport to the laboratory ( ⁇ 24 hours) . The sea-weed was then either dried immediately or stored in frozen state until further processing. For enzyme purification the material was stored at -18°C, whereas the material intended for isolation of mRNA was stored in liquid nitrogen.
• the sea-weed material was subjected to repeated extractions which were carried out as the first one described above.
• the material was usually discarded after 5-8 extractions when residual activity had declined to an almost negligible level.
• the filtrate was clarified by centrifugation at 10,000 x g in a Sorvall GSA rotor (Sorvall Instruments) .
• the supernatant was filtered through Whatman chromatography paper (chr 1) and diluted with water to a conductivity of 7-8 mS/cm. pH was adjusted to 7.5.
• the extract was then ready for anion exchange chromatography as described below.
• the assay mixture consisted of 1-40 ⁇ l of enzyme sample and 850 ⁇ l of an assay solution containing 370 ⁇ l of 0.1 M sodium phosphate buffer, pH 7.0; 462 ⁇ l of 0.1 M D-glucose in 0.1 M sodium phosphate buffer, pH 7.0; 9 ⁇ l of horse radish peroxidase, 0.1 mg/ml in water (Sigma Chemicals, cat. no. P 6782 or Boehringer Mannheim, cat. no. 814 393) ; and 9 ⁇ l of o-dianisidine-2HC1, 3.0 mg/ml in water (3,3' -dimethoxybenz- idine, Sigma Chemicals) .
• This step was carried out on a BioPilot chromatography system (Pharmacia Biotech, Sweden) connected to a SuperRac fraction collector (LKB-Adapter AB, Sweden) .
• XX420LCS0 XX420LCS0
• the system was equipped with a 30,000 nominal molecular weight limit (NMWL) membrane cell (cat. no. PTTKOLCP2) and was driven by a peristaltic pump. After con ⁇ centration at room temperature to about 50 ml, the enzyme preparation was further concentrated to 10-20 ml by centrifu ⁇ gal ultrafiltration at 4°C in Centriprep concentrators (Ami- con, USA, nominal molecular weight cut-off 30,000) according to the instructions of the manufacturer. The concentrated enzyme solution was stored at 4°C.
• NMWL nominal molecular weight limit
• composition of the preparation of hexose oxidase obtained by ion exchange chromatography and ultrafiltration was ana ⁇ lyzed by native PAGE on a Pharmacia Phast System, see Fig. 2.
• the 8-25% gradient gels were run and silver stained for protein according to the instructions of the manufacturer.
• a kit containing the following molecular weight markers was also obtained from Pharmacia: Thyreglobulin (669,000); ferritin (440,000); catalase (232,000); lactate dehydrogenase (140,000) and albumin (67,000).
• Phast gel was submerged in 10 ml of freshly prepared staining solution containing: 0.1 M D-glucose (or D-galactose) ; 85 mM citric acid/sodium phosphate pH 6.5; 0.2 mg/ml of 3- (4,5-dimethylthiazol-2-yl) - 2,5-diphenyl-tetrazolium bromide ("thiazolyl blue", MTT, Sigma Chemicals, cat. no. M 2128); and 0.1 mg/ml of N-methyl- dibenzopyrazine methyl sulfate salt ("phenazin methosulfate", PMS, Sigma Chemicals cat. no. P 9625) .
• the gel was incubated at room temperature in the dark until the coloured, blue- violet band was clearly visible (usually 5-90 minutes) and was then rinsed in 10% acetic acid, 5% glycerol and air- dried.
• the silver stained gel is shown in Fig. 2, lane 1. As it appears from the figure, numerous proteins were present at this step of the purification. By enzyme staining, however, only the band marked with an arrow in Fig. 2 was stained, (results not shown) .
• This step in the purification was carried out on an FPLC system (Pharmacia) equipped with a XK26/70 column (2.6 x 66 cm, Pharmacia) with a bed volume of 350 ml.
• the column was packed with Sephacryl S-200 HR (Pharmacia) according to the instructions of the manufacturer.
• the buffer was 20 mM Tris- Cl, 500 mM NaCl, pH 7.5 and the flow rate was 0.5 ml/min.
• the UV-absorbance at 280 nm was recorded. Fractions of 2.5 ml were collected with a FRAC-100 fraction collector (Pharmacia) which was placed in a refrigerator (4°C) next to the FPLC.
• the concentrated preparation of hexose oxidase was clarified by centrifugation at 30,000 rpm in a SW60 swinging bucket rotor (Beckman) in an L7 ultracentrifuge (Beckman) for 60 min at 4°C. An aliquot of 3.0-4.0 ml of the supernatant was mixed with 5% glycerol (Sigma Chemicals, cat. no. G 7757), filtered through a disposable filter unit with 0.22 ⁇ m pore size (Millipore, cat. no. SLGV 025 BS) and applied onto the column using an SA-5 sample applicator (Pharmacia) connected to the inlet of the column.
• glycerol Sigma Chemicals, cat. no. G 7757
• the molecular weight of native hexose oxidase was determined by gel filtration on Sephacryl S-200 Superfine (Pharmacia) . Column dimensions, buffer, flow rate and fraction collection were as described above. Blue dextran for determination of the void volume (v Q ) of the column and the following standard proteins for calibration of the column were obtained from Pharmacia: Ovalbumin (43,000), albumin (67,000), catalase
• This step was carried out on a SMART Micropurification Chro ⁇ matography System (Pharmacia) equipped with a HR5/5 column (Pharmacia, 0.5 x 5 cm, bed volume 1.0 ml) packed with S- Sepharose Fast Flow (Pharmacia) .
• the column was equilibrated in A-buffer: 50 mM sodium acetate, pH 4.5 (prepared by adjus- ting 50 mM acetic acid to pH 4.5 with NaOH) .
• the buffer B used for gradient elution contained 50 mM sodium acetate, 500 mM NaCl, pH 4.5.
• hexose oxidase was analyzed by native PAGE and silver staining (Fig. 2, lane 3) .
• the hexose oxidase band was now the only significant band, although small amounts of contaminating proteins were also observed.
• Fig. 4, lane 1 The result of the electrophoresis is shown in Fig. 4, lane 1.
• the purified preparation of hexose oxidase showed strong bands at relative molecular weights of 40 kD and 29 kD, respectively and faint bands at 60 kD and 25 kD, respective ⁇ ly. Furthermore, two sharp doublet bands at 55 kD and 57 kD were observed. 1.11. SDS-PAGE followed by blotting and staining for carbo ⁇ hydrate
• hexose oxidase from cation exchange chromatography was run on a 12% SDS-PAGE gel as described above, blotted to nitrocellulose according to standard procedures and stained for carbohydrate with the Glycan Detection Kit according to the instructions of the manufacturer. None of the hexose oxidase bands at 60 kD, 40 kD, 29 kD and 25 kD were stained. Only the sharp doublet band at 57 kD-55 kD was intensely stained (results not shown) . The 57 kD-55 kD doublet band was later identified as a residual contaminant as described below.
• Hexose oxidase fractions from S-Sepharose chromatography were pooled and concentrated by centrifugal ultrafiltration in
• Centricon concentrators (Amicon) and analyzed by isoelectric focusing (IEF) on Isogel agarose plates, pH 3-10, according to the instructions of the manufacturer (FMC Bioproducts, Rockland, ME, USA) .
• FMC Bioproducts A mixture of pi markers (FMC Bioproducts) were run in parallel with the hexose oxidase samples.
• the gels were stained with Coomassie Brilliant Blue R-250.
• lane 1 the purified preparation of hexose oxidase was composed of two variants with pi's of 4.3 and 4.5, respectively.
• Purified hexose oxidase was also analyzed by isoelectric focusing on pre-cast polyacrylamide gels, pH 3.5-9.5 (Pharmacia, Ampholi- ne PAGplates) according to the instructions of the manufac ⁇ turer. These gels were stained for enzyme activity by incuba ⁇ tion in a staining mixture as described above for native polyacrylamide gels.
• Fig. 5, lane 2 both pi variants were enzymatically active.
• Chromatofocusing was carried out on the SMART chromatography system equipped with a Mono P HR 5/5 column (0.5 x 5 cm, Pharmacia) with a bed volume of 1 ml and a 50 ml Superloop for sample application.
• the start buffer for separation in the interval between pH 5.0 and 3.5 was 25 mM piperazine adjusted to pH 5.5 with HCl.
• the eluent was Polybuffer 74
• Sample preparation was carried out in the following manner: In a typical experiment the best fractions from two gel filtration runs (2 x 4 fractions, 20 ml) were pooled and passed through a column of 1 ml of Phenyl Sepharose 6 Fast Flow (high sub, Pharmacia) which had been packed in a dispos ⁇ able Poly-prep column (Bio-Rad) and equilibrated in the buffer used for gel filtration (20 mM Tris-Cl, 500 mM NaCl, pH 7.5) .
• Hexose oxidase purified by chromatofocusmg was analyzed by native PAGE and silver staining (Fig. 2, lane 4) and by SDS- PAGE and staining with Coomassie Brilliant Blue (Fig. 4, lane 2) .
• native PAGE the hexose oxidase band was the only significant band, and only very low amounts of contaminants were observed.
• SDS-PAGE it was clearly demonstrated that this purification method was able to remove the sharp doublet band at 57 kD and 55 kD. The band at 25 kD observed after S- Sepharose chromatography was very faint after chromatofocus ⁇ mg.
• hexose oxidase obtained by DEAE chromato ⁇ graphy, gel filtration and chromatofocusmg showed one band in native PAGE.
• SDS-PAGE strong bands at 40 kD and 29 kD and a weak band at 60 kD were observed. Since the intensity of the 60 kD component, relative to the 40 kD and 29 kD components, varied between different prepara ⁇ tions of the enzyme, it was hypothesized that the 29 kD and 40 kD polypeptides might originate from proteolytic proces- sing of an about 60 kD precursor.
• Hexose oxidase obtained by purification on DEAE Sepharose, Sephacryl S-200, and S-Sepharose was transferred to a vola ⁇ tile buffer by buffer-exchange on a pre-packed PC3.2/10 Fast Desalting Column containing Sephadex G-25 Superfine (Pharma ⁇ cia, 0.32 x 10 cm, bed volume 0.8 ml) which was mounted in the above SMART system.
• the column was equilibrated and eluted with 200 mM ammonium bicarbonate (BDH, AnalaR) . To obtain a satisfactory recovery it was necessary to add 500 mM sodium chloride to the hexose oxidase sample before injec ⁇ tion.
• the peptides generated by cyanogen bromide digestion were separated by high resolution SDS-PAGE according to Sch gger & von Jagow (1987) . This system provides excellent separation of low molecular weight peptides (20-250 amino acid resi ⁇ dues) .
• the gel system consisted of a 16.5% separation gel, a 10% spacer gel and a 4% stacking gel, all made using a 29:1 acrylamide/bisacrylamide mixture from Promega.
• Minigels with a thickness of 0.75 mm were run in a Mini- Protean II apparatus (Bio-Rad) .
• Ammonium persulfate and N,N,N' ,N' -tetramethyl-ethylenediamine (TEMED) were from Bio- Rad.
• SDS was from United States Biochemical (ultrapure) .
• Tris was from Fluka (cat. no. 93350) .
• Tricin and sodium thiogly- cate were from Promega. Glycin (p.a.), 2-mercaptoethanol
• bromophenol blue was from Merck and glycerol from GIBCO BRL (ultrapure) .
• the gel was pre-run for 60 min at 30 V to allow the thioglycolate to scavenge any amino-reactive substances.
• Sample preparation The dried cyanogen bromide peptide frag ⁇ ments were resuspended in 30 ⁇ l of gel loading buffer con ⁇ taining 63 mM Tris-Cl, pH 6.8, 1% SDS, 2.5% 2-mercapto- ethanol, 10% glycerol and 0.0012% bromophenol blue. Samples that turned yellow upon mixing, due to the content of resi ⁇ dual formic acid were neutralized by addition of 1-3 ⁇ l of 1.0 M Tris base until the blue colour was restored. The samples were denatured by heating at 95°C for 5 min before application on the gel.
• Electrophoretic transfer to PVDF membrane was carried out in a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) according to the instructions of the manufacturer. Three sheets of Problott membrane (Applied Biosystems) cut to the size of the gel were wetted briefly in methanol (Merck, p.a) and then soaked in transfer buffer (25 mM Tris, 192 mM glycine, pH 8.5, pre-cooled to 4°C) until assembly of the blotting sandwich.
• transfer buffer 25 mM Tris, 192 mM glycine, pH 8.5, pre-cooled to 4°C
• the gel was incu ⁇ bated in transfer buffer for 5 min at 4°C and then assembled into a transfer sandwich having the following layers: A sheet of Whatman paper (3MM chr) , two sheets of Problott membrane, the SDS-PAGE peptide separation gel, the third sheet of Problott, and a final sheet of Whatman paper.
• the sandwich was oriented with the two sheets of Problott membrane toward the positive electrode in the electrode assembly.
• the cooling unit was mounted in the buffer chamber before it was filled with pre-cooled transfer buffer, and the transfer was then performed at room temperature for 60 min at 100 V constant voltage. During transfer the current increased from about 270 mA to about 400 mA.
• the membrane was washed in water for 1 min and then stained for 30-45 sec in 100 ml of freshly prepared staining solution containing 0.1% Coomassie Brilliant Blue R- 250 (Bio-Rad) , 5% acetic acid (Merck, p.a) and 45% methanol (Merck, p.a) .
• the membrane was then destained with 3 changes of about 80 ml of freshly prepared 5% acetic acid, 45% meth- anol for 30-60 sec each.
• the membrane was finally washed in 3 changes of water to remove residual glycine and then air- dried.
• Well-resolved and relatively abundant bands of mo ⁇ lecular weights of about 2.5 kD, 9 kD and 16 kD, respective- ly were excised and submitted to amino acid analysis and sequence analysis.
• Amino acid analysis was carried out by ion exchange chroma- tography and post-column derivatization with o-phtaldialde- hyde. Samples were hydrolyzed at 110°C for 2Oh in 6 M HCl, 0.05% phenol and 0.05% dithiodipropionic acid (Barkholt and Jensen, 1989) . Peptides were sequenced on an automated pro- tein/peptide sequencer from Applied Biosystems, model 477A, equipped with on-line PTH analyzer, model 120A and data analysis system. Protein sequencing reagents were obtained from Applied Biosystems. Amino acid analysis and peptide sequence analysis was kindly performed by Arne L. Jensen, Department of Protein Chemistry, University of Copenhagen, Denmark.
• the peptide sequence identified by analysis of the 9 kD fragment is shown in Table 2.1.
• the initial yield of phenylthiohydantoin-tyrosine (PTH-Tyr) at step one was 22 pmol.
• the amino acid composition of the 9K fragment is shown in Table 2.2.
• Sample preparation Fractions from chromatofocusing were concentrated by centrifugal ultrafiltration at 4°C in Ultra- free-MC filter units with NMWL 10,000 and a sample capacity of 400 ⁇ l (Millipore, cat. no. UFC3 LGC25) .
• the retentate was mixed with one volume of gel loading 2X buffer containing 125 mM Tris-Cl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 20% glycerol and 0.0025% bromophenol blue. Samples that turned yellow upon mixing, due to the content of acidic Polybuffer components, were neutralized by addition of 1-3 ⁇ l of 1.0 M Tris base until the blue colour was restored.
• the samples were denatured by heating at 95°C for 5 min and applied on the gel in aliquots of about 30 ⁇ l per lane.
• a mixture of molecular weight marker proteins (Bio-Rad) with molecular weights ranging from 97,400 to 14,400 was run in parallel with the hexose oxidase samples.
• the electrophoresis was run at low current, 10 mA per gel, in order to minimize the risk of thermally induced, chemical modification of the sample proteins.
• Electrophoretic transfer to PVDF membrane was carried out as described above, except that one sheet of Immobilon P Mem ⁇ brane (Millipore, cat. no. IPVH 15150) was used instead of three sheets of Problott.
• the sandwich was oriented with the blotting membrane toward the positive electrode in the elec ⁇ trode assembly.
• the Immobilon P membrane was rinsed in water for 10 sec and then stained for 45-60 sec in 100 ml of fresh ⁇ ly prepared staining solution containing 0.025% Coomassie Brilliant Blue R-250, 5% acetic acid and 40% methanol. The membrane was then destained for 2-3 min in 250 ml of freshly prepared 5% acetic acid, 30% ethanol (96% v/v, Danisco,
• the band pattern on the blot w ⁇ s identical to the pattern seen in analytical SDS-PAGE after final purification by chromatofocusing. It showed strong bands at 40 kD and 29 kD, in addition to a faint band at 60 kD.
• the proteolytic enzyme selected for the digestion was endoproteinase Lys-C (endoLys-C) which cleaves peptide chains at the C-terminal side of lysine residues.
• EndoLys-C endoproteinase Lys-C
• An aliquot of 5 ⁇ g of endoLys-C (Boehringer Mann ⁇ heim, sequencing grade, cat. no. 1047 825) was reconstituted by addition of 20 ⁇ l of water.
• Two ⁇ l of enzyme solution, corresponding to 0.5 ⁇ g was added (enzyme:substrate ratio 1:10) . Digestion was carried out at 37°C for 22-24 h.
• the peptide fragments obtained by digestion of 40 kD and 29 kD polypeptides of hexose oxidase were separated on the SMART chromatography system.
• the system was equipped with a variable-wavelength ⁇ Peak monitor and a fraction collector bowl for 60 vials.
• the reversed phase column used for the separation was a silica-based ⁇ RPC C2/C18 SC2.1/10 narrow- bore column (Pharmacia, column dimensions 2.1x100 mm, par ⁇ ticle size 3 ⁇ m, average pore size 125 A) .
• the buffers were A: 0.1% TFA (Pierce) in Milli-Q water and B: 0.1% TFA in acetonitrile (Merck, gradient grade Lichrosolv) .
• the buffers were filtered and degassed by vacuum filtration on a 0.5 ⁇ m fluoropore filter (Millipore, cat. no. FHLP04700) .
• the flow rate was 100 ⁇ l/min.
• UV-absorbance in the effluent was moni ⁇ tored at 220 nm, 254 nm and 280 nm.
• the gradient was 0-30% B (0-65 min), 30-60% B (65-95 min) and 60-80% B (95-105 min).
• the reason for this might be that hydrogenated Triton X-100 from Calbiochem was used for the 29 kD digestion, whereas the 40 kD digestion was carried out with Triton X-100 from Sigma Chemicals) .
• the peptide sequences identified by analysis of fractions corresponding to peaks 1-5 in Fig. 6 (HOX-2, HOX-3, HOX-4, HOX-5 and HOX-6 peptides) and peaks 1-2 in Fig. 7 (HOX-7 and HOX-8 peptides) are shown in the below Table 2.4.
• the initial yields of PTH amino acids ranged from 46 pmol of PTH-Tyr at step one in the HOX-5 peptide to 6 pmol of PTH-lie at step two in the HOX-8 peptide.
• As expected from the absorbances at 254 nm and 280 nm, respectively of the selected peaks all the sequenced peptides contained at least one aromatic amino acid residue.
• Residue no. 15 was identified as either Asp or Asn.
• Residue no. 17 was identified as either Arg or Trp.
• HOX-3 peptide SEQ ID NO:10
• HOX-4 peptide SEQ ID NO:11
• HOX-5 peptide SEQ ID NO:12
• HOX-6 peptide SEQ ID NO:13
• HOX-7 peptide SEQ ID NO:14
• Freshly collected fronds of Chondrus crispus were rinsed with cold water and immediately stored in liquid nitrogen until further use. About 15 grams of Chondrus crispus thallus frozen in liquid nitrogen was homogenized to a fine powder in a mortar. The frozen, homogenized material was transferred to a 50 ml tube ( ⁇ unc, cat. no. 339497) containing 15 ml extrac- tion buffer (8M guanidinium hydrochloride; 20 mM 2-( ⁇ -morpho- lino)ethanesulfonic acid (MES) , pH 7.0; 20 mM ethylenedi ⁇ aminetetraacetic acid (EPTA) ; 50 mM jS-mercaptoethanol) .
• extrac- tion buffer 8M guanidinium hydrochloride; 20 mM 2-( ⁇ -morpho- lino)ethanesulfonic acid (MES) , pH 7.0; 20 mM ethylenedi ⁇ aminetetraacetic acid
• the tube was vortexed and kept cold (0°C) during the follow ⁇ ing steps unless other temperatures are indicated. Then the tube was centrifuged for 20 minutes at 6,000 x g in a Heraeus Omnifuge 2.0RS and the RNA-containing supernatant (about 15 ml) was carefully collected and transferred to a pre-chilled 50 ml tube. 1.5 ml 2 M sodium acetate, pH 4.25, 15 ml water saturated phenol and 3 ml chloroform:isoamyl alcohol (49:1) was added to the tube containing the RNA extract.
• the tube was subsequently vortexed vigorously for 1/2 minute and the phases were separated by ce ' ntrifuging the tube for 20 minutes in an Omnifuge at 6,000 x g.
• the aqueous phase (about 17 ml) was transferred to a 30 ml Corex tube (Sorvall, cat. no. 00156) and an equal volume (i.e. about 17 ml) of cold isopropanol was added.
• the tube was vortexed again and incu ⁇ bated for at least 1 hour at -20°C.
• the precipitated RNA was pelleted by centrifugation for 20 minutes at 10,000 rpm using a Sorvall RC-5B centrifuge provided with a pre-chilled SS: :• rotor.
• RNA pellet was precipi ⁇ tated on ice for 30 minutes and pelleted again as described above.
• the RNA pellet was washed in 70% ethanol and resuspen ⁇ ded in 500 ⁇ l water. The resuspended RNA was transferred to a microcentrifuge tube and stored at -20°C until further use.
• RNA purity and concentration of the RNA was analyzed by agarose gel electrophoresis and by absorption measurements at 260 nm and 280 nm as described in Sambrook et al. (1989) .
• Poly-adenylated RNA was isolated from total RNA using magne ⁇ tic beads containing oligo dT (Pynabeads ® Oligo (dT) 25 , in mRNA Purification KitTM, Pynal) . Approximately 100 ⁇ g total RNA was mixed with 1 mg Pynabeads ® Oligo (dT) 25 and poly- adenylated RNA was isolated as described in the protocol for the mRNA Purification KitTM. The yield of poly-adenylated RNA isolated with Pynabeads ® was between 1 and 3%.
• RNA Separator KitTM oligo- (dT) -cellulose
• RNA isolated on oligo- (dT) columns compared to Pynabeads ® purified RNA could be the presence of carbohydrates or pro- teoglycans in the extract of total RNA.
• Carbohydrates con ⁇ taminating the total RNA preparations have been shown to impede the purification of poly-adenylated RNA and to inhibit cPNA synthesis and therefore, methods for the purification of RNA free of carbohydrates have been developed (Groppe et al.
• poly-adenylated RNA purified with these methods was not as effective in cPNA synthesis reac ⁇ tions as RNA isolated with Pynabeads ® . Accordingly, poly- adenylated RNA purified using Pynabeads ® was used as template in first strand cPNA synthesis reactions (cf. 3.4 below).
• Synthetic oligonucleotides were synthesized (PNA technology, ApS, Forskerparken, PK-8000 Aarhus C, Penmark) based on the amino acid sequences derived from hexose oxidase peptides HOX-2, HOX-3 and HOX-4 (Table 2.4).
• Table 3.1 shows the oligonucleotides and their corresponding amino acid sequences. Also shown in Table 3.1 is the PNA sequence of the primers used in PNA sequencing or in PCR.
• Poly-adenylated RNA was used as template in first strand cPNA synthesis reactions with commercially available kits. About 1 ⁇ g poly-adenylated RNA was reverse transcribed as described in the protocol for MaratonTM cPNA Amplification Kit (Clon ⁇ tech, cat. no. K1802-1) with Hox3-2- or Hox4-2- as primers. In the subsequent PCR amplification the anchor or adaptor primer of the kit was used in addition to the hexose oxidase specific primers Hox3-2- or Hox4-2-, respectively. The buf ⁇ fers used and the conditions for amplification was essential ⁇ ly as described in the protocol for the MaratonTM cPNA Ampli ⁇ fication Kit.
• PCR amplification was carried out with AmpliTaq (Perkin-Elmer Cetus) using a Perkin-Elmer Thermalcycler 480TM programmed to 30 cycles at 1 min at 94°C, 2 min at 55°C and 2 min at 72°C.
• Gel electrophoresis of 5 ⁇ l of the reaction mixture in a 1% agarose gel showed PNA fragments with approximate sizes of 600 base pairs (bp) with primer Hox4-2- and of 700 bp with primer Hox3-2-.
• PNA fragments were purified from the agarose gel using a commercially available kit (QIAEXTM Gel Extraction Kit, cat. no. 20020, QIAGEN) and about 100 ng fragment was ligated to 50 ng plasmid pT7 Blue as described in the protocol for pT7 Blue T-Vector Kit (Novagen, cat. no. 69829-1) . Escherichia coli PH5o. (Life Techiv.ilogies, cat. no. 530-8258SA) or E. coli NovaBlue (Novagen) was transformed with the ligation mixture, and white, recombinant colonies were analyzed further.
• QIAEXTM Gel Extraction Kit cat. no. 20020, QIAGEN
• Plasmid PNA from such colonies was purified using QIAGEN Plasmid Midi Kit (QIAGEN, cat. no. 12143) and subjected to PNA sequence analysis using Sequenase (Sequenase Version 2.0 PNA Sequencing Kit, USB) .
• PNA sequencing reactions were subjected to acrylamide gel electrophoresis (Sequencing Gel- Mix ® 6, Life Technologies) .
• PNA sequence analysis of the 700 bp fragment showed an open reading frame with a coding capa ⁇ city of 234 amino acids.
• Table 3.2. shows that all the peptide sequences from the 40 kP polypeptide, i.e. HOX-2, HOX-3, HOX-4, HOX-5, and HOX-6, were found in the 234 amino acid sequence.derived from this open reading frame. Thus, it was concluded that the 700 bp fragment encoded part of the hexose oxidase gene.
• the PNA sequence of the 600 bp fragment was shown to be identical to the proximal 600 bp of the 700 bp fragment (see Table 3.2.).
• Primers Hox2-3+ and Hox3-2- were used similarly in cPNA synthesis and PCR amplification experiments. About 50 ng poly-adenylated RNA was reverse transcribed with Hox3-2- as primer as described in the protocol for 3' -AmplifinderTM RACE Kit (Clontech, cat. no. K1801-1) . In the subsequent PCR amplification primers Hox2-3+ and Hox3-2- were used. The buffers used and the conditions for amplification were essen ⁇ tially as described for AmpliTaq polymerase (Perkin-Elmer Cetus) and in the protocol for 3' -AmplifinderTM RACE Kit. Gel electrophoresis of 5 ⁇ l of the PCR amplification mixture showed a fragment with a size of 407 bp.
• This fragment was purified, inserted into plasmid pT7 Blue and sequenced as described above.
• the PNA sequence of this fragment was shown to be identical to the distal 407 bp of the 700 bp fragment.
• the PNA sequence downstream of the 700 and 407 bp fragments was amplified with the 3'AmplifinderTM RACE Kit (Clontech) using the anchor primer of the kit as 3'primer and the hexose oxidase specific primers Hox5+ and Hox4+ as gene specific 5' primers.
• the buffers and the conditions for amplification were as described above.
• PCR and analysis of the reaction mixture on agarose gels showed a fragment with the size of about 1.3 kb.
• the fragment was isolated and subjected to PNA sequence analysis as described above.
• the PNA sequence of this 1.3 kb fragment showed an open reading frame of 357 amino acids.
• This 357 amino acid reading frame contained the amino acid sequences of the peptides HOX-1, HOX-3, HOX-4, HOX-5, HOX-7 and HOX-8. Therefore, it was concluded that the 1.3 kb DNA fragment encoded the 9 kP CNBr fragment, the 29 kP polypeptide, and part of the 40 kP polypeptide of hexose oxidase.
• the gene was amplified using PCR, inserted into pT7 Blue and sequenced as described above.
• the PNA sequence of this 1.8 kb fragment was identical to the PNA sequences of the fragments described above with minor differences. Since these differences could be caused by misincorporations during PCR amplifications, the entire hexose oxidase gene was amplified and isolated from at least three independent PCR amplifications. Therefore, the DNA sequence presented in the below Table 3.2. is composed of at least three independently derived PNA sequences in order to exclude PCR errors in the sequence.
• the amino acid sequence derived from the open reading frame on the above 1.8 kb PNA sequence is shown to contain all of the above HOX peptides, ie HOX-1 to HOX-8. Accordingly, the 1.8 kb PNA sequence codes for the above 9 kP, 29 kP and 40 kP Chondrus crispus-derived hexose oxidase fragments.
• the mole ⁇ cular weight of this derived open reading frame polypeptide is consistent with the assumption that the polypeptide is a subunit (possibly a monomeric fragment) of a dimeric hexose oxidase enzyme molecule.
• RNA isolated from Chondrus crispus was subjected to Northern blot analysis.
• RNA was purified as described above (3.1) and fractionated on a denaturing formaldehyde agarose gel and blotted onto a HybondC filter (Amersham) as described by Sambrook et al. (1989) .
• a 400 bp PNA fragment was synthesized by PCR as described above (3.4).
• This fragment was purified from a 1.2% agarose gel (SeaPlaque ® GTG, FMC) and labelled with 32 P as described by Sambrook et al. ( supra) .
• This radioactive hexose oxidase specific hybridization probe was used to . probe the Northern blot.
• the conditions for hybridization was:
• the filter was washed twice at 65°C for 10 minutes in 2 x SSC, 0.1% SPS followed by two washes at 65°C for 10 min in 1 x SSC, 0.1% SPS.
• ATC GTC TCT GGC GGC CAT TGC TAC GAG GAC TTC GTA TTT GAC GAA TGC 350 lie Val Ser Gly Gly His Cys Tyr Glu Asp Phe Val Phe Asp Glu Cys 75 80 85
• the HOX-l to HOX-8 peptides are shown with bolded or underlined codes.
• Bolded codes indicate amino residues which have been confirmed by amino acid sequencing of the peptides.
• the underlined codes indicate amino acid residues which are derived from the nucleotide sequence, but which have not been confirmed by sequencing of the relevant HOX peptides.
• HOX-l is amino acid residues 461-468, HOX-2 residues 92-114, HOX-3 residues 219-234, HOX-4 residues 189-202, HOX-5 resi- dues 215-218, HOX-6 residues 8-22, HOX-7 residues 434-444 and HOX-8 residues 452-460.
• the plasmid contains the alcohol oxidase promotor (aoxl promotor) and transcriptional termination signal from Pichia pastoris (in Fig. 9, aoxp and aoxt, respectively) .
• a his4 + gene in the vector enables selection of His + recombinant Pichia pastoris cells.
• this expression cassette When this expression cassette is transformed into Pichia pastoris it integrates into the chromosomal PNA.
• Pichia pastoris cells harbouring an expression cassette with a Chondrus crispus hexose oxidase gene inserted downstream of the aoxl promotor can be induced to produce hexose oxidase by the addition of the inducer of the aoxl promotor, methanol.
• a mutant of Pichia pastoris, KM71, which is defective in the major alcohol oxidase gene, aoxl can be used as recipient of the hexose oxidase gene (Cregg and Madden 1987; Tschopp et al. 1987) .
• Pichia pastoris contains another alcohol oxidase gene, aox2, which can also be induced by methanol.
• recombinant Pichia pastoris transformed with a hexose expression cassette will produce two oxidases, hexose oxidase and alcohol oxidase, upon addition of methanol.
• hexose oxidase gene Before insertion of the hexose oxidase gene into the expres ⁇ sion vector pPIC3, sequences 5' and 3' of the open reading were modified.
• First strand cPNA was used as template in PCR.
• the synthetic oligonucleotide specific for the 5'-end of the open reading frame, Hox5'-l (Table 3.1) was used as PCR- primer together with a primer (Hox3'-l) specific for the 3'- end of the sequence encoding Chondrus crispus hexose oxidase.
• the primer Hox3'-l had the sequence 5'-ACCAAGTTTATAAAAAGCAACCATCAC-3' (SEQ IP NO:32).
• PCR amplifi ⁇ cation was carried out using the GeneAmp PCR Reagent Kit with AmpliTaq PNA polymerase (Perkin-Elmer Cetus) .
• the PCR pro ⁇ gram was 30 cycles at 30 sec at 94°C, 30 sec at 55°C and 2 min at 72°C.
• Gel electrophoresis of the reaction mixture showed a band with the approximate size of 1.7 kb. This 1.7 kb fragment was inserted into the vector pT7 Blue (Novagen) (plasmid pUPO150) and subjected to PNA sequencing.
• the P ⁇ A was restricted further with BcoRI and the P ⁇ A fragment containing the hexose oxidase gene was purified on an agarose gel as a blunt end - EcoRI P ⁇ A fragment (QIAEXTM, QIAGEN) .
• the Pichia pastoris expression vector pPIC3 was restricted with the restriction enzymes SnaBI and EcoRI and purified on an agarose gel. The purified vector and the fragment encoding hexose oxidase were ligated and the ligation mixture was transformed into JB. coli PH5o. (Life Technologies) , essential- ly as described by Sambrook et al. (1989). The resulting expression vector containing the hexose oxidase gene from Chondrus crispus, plasmid pUP0153, was subjected to PNA sequencing to ensure that no mutations had occurred in the hexose oxidase gene during the subcloning procedure.
• Plasmid pUP0153 was purified from E. coli PH5o. and introduced into Pichia pastoris using electroporation (The Pichia Yeast Expression System, Phillips Petroleum Company) or using The Pichia Spheroplast Module (Invitrogen, San Piego, USA, cat. no. K1720-01) .
• Pichia pastoris strain KM71 containing the expression cas ⁇ sette with the hexose oxidase gene inserted between the aoxl promoter and the transcription termination signal was culti ⁇ vated in shake flasks in MP (1.34 grams per liter of yeast nitrogen base (Pifco, cat. no. 0919-15-3), 0.4 mg/1 of bio ⁇ tin, 0.1% arginine, and 20 g/1 glucose) .
• One-liter shake flasks containing 150 ml culture were incubated in a rotary shaker at 30°C, 300 rpm.
• the cells were harvested by centrifugation (6,000 x g, 10 min) and resuspended in about 1/5 of the growth volume of 50 mM Tris-Cl, pH 7.5. Resuspended cells were kept cold until disrupture in a FRENCH Press (SLM Instruments, Inc., Rochester, N. Y.). Cells were opened in a 20K FRENCH Pressure Cell at an inter ⁇ nal pressure of 20,000 psi. The cell extract was cleared by centrifugation at 10,000 x g for 10 min at 5°C. The hexose oxidase containing supernatant was carefully removed and subjected to purification as described below.
• Clarified homogenate from FRENCH press homogenization (100- 150 ml) was subjected to anion exchange chromatography on an FPLC system equipped with two 5-ml HiTrap-Q columns prepacked with Q-Sepharose High Performance (Pharmacia) .
• the columns were connected in series and the chromatography was carried out at room temperature.
• the column was equilibrated in buffer A: 20 mM Tris-Cl, pH 7,5.
• the flow rate was 1.25 ml during sample application and 2.5 ml during wash and elution. After sample application the column was washed with 30 ml of buffer A.
• Adsorbed proteins were then eluted with 200 ml of a gradient from buffer A to buffer B: 20 mM Tris-Cl, 750 mM NaCl, pH 7.5. Fractions of 2 ml were collected during wash and gradient elution. The fractions were assayed for hexose oxidase activity as described above in Example 1.3 (10 ⁇ l of sample, 15 min of incubation time) . The fractions were also assayed for alcohol oxidase (AOX) activity in an assay which was identical to the hexose oxidase assay except that 0.5% methanol instead of 0.05 M glucose was used as substrate.
• AOX alcohol oxidase
• the activity profiles showed that AOX and HOX co-eluted at a salt concentration of about 400 mM NaCl. Fractions containing hexose oxidase were pooled and stored at 4°C.
• Second step gel filtration.
• the pool from step one in the purification (20-30 ml) was concentrated to about 3.5 ml by centrifugal ultracentrifuga- tion at 4°C in Centriprep concentrators (Amicon, USA, nomi ⁇ nal molecular weight cut-off 30,000).
• the concentrated prepa ⁇ ration of hexose oxidase was clarified by centrifugation and the supernatant was mixed with glycerol to a final concentra- tion of 5%.
• the sample was applied onto the column using an SA-5 sample applicator (Pharmacia) connected to the inlet of the column.
• the pool from the above second step was further purified by anion exchange chromatography on a FPLC system equipped with a Mono Q HR 5/5 column (bed volume 1 ml) .
• the column was equilibrated in buffer A: 20 mM Tris-Cl, pH 7,5.
• the flow rate was 1 ml/min.
• the pool from step two was desalted by gel filtration in buffer A on pre-packed Sephadex G-25 columns (PP-10, Pharmacia) . After sample application the column was washed with 30 ml of buffer A.
• Adsorbed proteins were eluted with 20 ml of a gradient from 0% to 100% buffer B: 20 mM Tris-Cl, 500 mM NaCl, pH 7,5.
• Fractions of 0,5 ml were col- lected and assayed for hexose oxidase activity as described above (10 ⁇ l of sample, 15 min of incubation time) . Fractions containing hexose oxidase were pooled and stored at 4°C.
• the pool from the above third step was purified by chromato- focusing on a Mono P HR 5/5 column as described above in Example 1.13, except that the Phenyl Sepharose adsorption step was omitted.
• the specific activity of recombinant hexose oxidase from Pichia pastoris was simi ⁇ lar to that of the native form isolated from Chondrus cris ⁇ pus.
• Fractions containing hexose oxidase were analyzed by SPS-PAGE and staining of the gel with Coomassie Brilliant Blue R-250 as described above.
• the purified preparation of recombinant hexose oxidase was composed of two bands migrat ⁇ ing at 40 kP and 29 kP, respectively.
• recombinant hexose oxidase could be isolated and purified from the host organism Pichia pastoris.
• SPS- PAGE the recombinant, purified enzyme exhibited the same bands at 40 kP and 29 kP as the corresponding native enzyme from Chondrus crispus.
• rHOX Purified rHOX was used for preparative SPS-PAGE and electro- blotting to PVPF membrane, as described above in Example 2.4.
• the resulting 40 kP and 29 kP bands were subjected to enzy ⁇ matic digestion of PVPF-bound hexose oxidase polypeptides, as described above in Example 2.5.
• the peptide fragments were separated by reversed-phase liquid chromatography as des ⁇ cribed above in Example 2.7.
• Well-resolved and abundant peptides were selected for amino acid sequence analysis by automated Edman degradation (10 steps) , as described above in Example 2.3. The obtained amino acid sequences are shown in Table 4.1.
• the HOX-9 peptide sequence from the recombinant 40 kP poly- peptide showed a sequence identical to Aspx 8 through
• Pichia pastoris transformed with the hexose oxidase gene from Chondrus cris ⁇ pus was capable of producing recombinant hexose oxidase.
• the substrate specificity of recombinant hexose oxidase from Pichia pastoris and native hexose oxidase from Chondrus crispus was compared using a number of sugars at a final concentration of 0.1 M in the assay described above. The relative rates are shown in Table 4.2.
• the open reading frame encoding Chondrus crispus hexose oxidase shown in Table 3.2. was .inserted into an Escherichia coli expression vector, pET17b (Novagen, cat. no. 69726-1) .
• the plasmid contains a strong inducible bac ⁇ teriophage T7 promotor and a T7 transcription termination signal. Genes inserted between these controlling elements can be expressed by the addition of isopropyl 5-P-thiogalactopy- ranoside (IPTG) if the plasmid is propagated in special E. coli host cells e.g. strain BL21(PE3) (Novagen, cat. no. 69387-1) .
• the hexose oxidase gene was modified at the 5' and 3' ends in order to insert the gene in the expression vector pET17b.
• the hexose oxidase gene was isolated by PCR with primers specific for the 5' and 3' ends of the hexose oxidase gene.
• the 5' primer (Hox5'-2) had the PNA sequence 5'- ATGAATTCGTGGGTCGAAGAGCCC-3' (SEQ IP NO:33) and the primer specific for the 3'-end was Hox3'-l.
• First strand cPNA from Chondrus crispus was used as template.
• PCR amplification was carried out with AmpliTaq PNA polymerase (Perkin-Elmer Cetus) as described in example 4.1. Gel electrophoresis of the reaction mixture showed a band with the approximate size of 1.7 kb. This 1.7 kb fragment was inserted into the vector pT7 Blue (Novagen) giving rise to plasmid pUPOl ⁇ l.
• the reaction mixture was fractionated on a 2 % agarose gel and the hexose oxidase specific 180 bp fragment was purified as described in Example 3.4.
• the 180 bp fragment was restricted with the restriction endonuclease Clal and EcoRl and ligated to pUP0161 restricted with the same enzymes giving rise to plasmid pUP0167.
• the hexose oxidase gene in plasmid pUP0167 was further sub- cloned in order to construct a hexose oxidase expression vector for E. coli .
• Plasmid pUP0167 was restricted with the enzymes Ndel and BazriHI and with the enzymes BamHI and Sail .
• the first reaction gave rise to a 1.6 kb fragment encoding the 5' and the middle part of the hexose oxidase gene while the reaction with the enzymes BamHI and Sail gave a 200 bp fragment encoding the 3' end of the hexose oxidase gene.
• Plasmid pET17b harbouring the hexose oxidase gene was denoted pUP0181. P ⁇ A sequencing showed that no mutation was introduced in the hexose oxidase gene during the isolation and cloning process.
• Plasmid pUPOl ⁇ l was introduced into E. coli strain BL2KPE3) (Novagen) by a standard transformation procedure (Sambrook et al. 1989) .
• the cells were grown in shake flasks in LB medium (Sambrook et al. supra) .
• IPTG 10 "3 M IPTG.
• One hour after the addition of IPTG the cells were harvested by centrifugation and resuspen ⁇ ded in sample buffer and subjected to SPS-PAGE as described above in Example 1.10.
• the result of the electrophoresis is shown in Figure 13.
• coli expressing recombinant hexose oxi ⁇ dase enzyme from plasmid pUPOl ⁇ l showed a prominent protein band at Mr 62 kP. This 62 kP band had the same molecular weight as the translation product predicted from the open reading frame. Non-transformed E. coli cells showed no such 62 kP protein.
• Plasmid pYES2 is a high-copy number episomal vector designed for inducible expression of recombinant proteins in Saccharomyces cerevisiae.
• the vector contains upstream acti ⁇ vating and promoter sequences from the S. cerevisiae Gall gene for high-level, tightly regulated transcription.
• the transcription termination signal is from the CYC1 gene.
• the hexose oxidase gene from Chondrus crispus was modified at the 5'- and 3'-ends in order to insert the gene in the ex ⁇ pression vector pYES2.
• the hexose oxidase gene was isolated from plasmid pUP0150 as described in Example 4.1 ( Figure 9) .
• the hexose oxidase gene was isolated on a blunt end-BcoRI PNA fragment as described and inserted into plasmid pYES2 re- stricted with the enzymes PvuII and EcoRI ( Figure 14) .
• the resulting plasmid, pUP0155 was subjected to PNA sequencing in order to show that no mutation had occurred during the cloning procedure.
• Plasmid pUP0155 was purified from E. coli PH5c- and trans ⁇ formed into S. cerevisiae by electroporation (Grey and Brend- el 1992) .
• the strain PAP1500 (genotype oc, ura3 -52, trpl : . - GAL10 -GAL4 , lys2 - 801 , leu2Al , his3 ⁇ 200, pep4::HIS3, prbl ⁇ l.6R, canl, GAL) (Pedersen et al. 1996) was used as a recipient.
• S. cerevisiae strain 1500 containing plasmid pUP0155 was grown and induced with 2% galactose as described by Pedersen et al. (1996) .
• Three days after the induction the cells were harvested by centrifugation and lysed as described above in Example 4.2.
• the crude extract was assayed for hexose oxidase activity using the o-dianisidine assay described above in Example 1.3.
• Table 6.1 shows that S. cerevisiae cells harbou- ring the hexose oxidase gene are capable of expressing active hexose oxidase.
• Hie indications nude below relate to the microorganism referred to in the description on page 66 . line 9
• the applicants request that a sample of the deposi ⁇ ted microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.
• the applicants request that a sample of the deposi ⁇ ted microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.
• the applicants request that a sample of the deposi ⁇ ted microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.
• Ala lie lie Asn Val Thr Gly Leu Val Glu Ser Gly Tyr Asp Xaa Xaa 1 5 10 15
• Ala lie lie Asn Val Thr Gly Leu Val Glu Ser Gly Tyr Asp Xaa Xaa 1 5 10 15
• Asp Pro Gly Tyr lie Val lie Asp Val Asn Ala Gly Thr Pro Asp 1 5 10 15
• MOLECULE TYPE DNA (genomic)
• FEATURE FEATURE:
• ATC GTC TCT GGC GGC CAT TGC TAC GAG GAC TTC GTA TTT GAC GAA TGC 350 lie Val Ser Gly Gly His Cys Tyr Glu Asp Phe Val Phe Asp Glu Cys 75 80 85
• Gly His lie Val Gly Gly Gly Asp Gly lie Leu Ala Arg Leu His Gly 145 150 155 160
• Phe Gly lie lie Thr Lys Tyr Tyr Phe Lys Asp Leu Pro Met Ser Pro 210 215 220
• Glu Gin lie Tyr Lys Thr Cys Glu Pro Thr Lys Ala Leu Gly Gly His 305 310 315 320
• Lys Ser Ala Tyr Met lie Lys Asp Phe Pro Asp Phe Gin lie Asp Val 370 375 380 lie Trp Lys Tyr Leu Thr Glu Val Pro Asp Gly Leu Thr Ser Ala Glu 385 390 395 400
• Thr Asn Lys Gin Ser lie Pro Thr Lys Pro Leu Lys Glu Pro Lys Gin 530 535 540

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